Technical Considerations for Scale-Up of Imine-Reductase-Catalyzed Reductive Amination: A Case Study

Imine reductases (IREDs) have attracted increasing attention as novel biocatalysts for the synthesis of various cyclic and acyclic amines. Herein a number of guidelines and considerations toward the development and scale-up of IRED catalyzed reactions have been determined based on the reductive amination of cyclo-hexanone (1) with cyclopropylamine (2). A Design of Experiments (DoE) strategy has been followed to study the different reaction parameters, facilitating resource-efficient and informative screening. Enzyme stability was identified to be the limiting factor. By moving from batch to fed-batch, it was possible to double the concentration of the substrate and turnover number (TON). Kinetic studies revealed that IRED-33 was the best enzyme for the reaction with respect to both activity and stability. Under the optimal reaction conditions, it was possible to react 1 and 2 at 750 mM concentration and reach 100% conversion to the desired amine (>90% isolated yield) in the space of 8 h. Hence, excellent volumetric productivity of 12.9 g L-1 h(-1) and TON above 48 000 were achieved.